A method of spatial high resolution imaging of a structure of a sample, the structure comprising a luminophore, is known as STED (Stimulated Emission Depletion) scanning fluorescence light microscopy. Here, the sample, in the measurement area, is at first subjected to the luminescence excitation light which excites the luminophore out of the excitable electronic ground state into the excited luminescent state. Then, the sample, in the measurement area, is subjected to an intensity distribution of the luminescence de-excitation light in the form of emission stimulation light which stimulates the luminophore for emission of light having the wavelength of the emission stimulation light, i.e. a different wavelength than that one of the luminescence light, and thus de-excites it back into its ground state, the intensity distribution having a local minimum. If the luminescence de-excitation light de-excites the luminophore out of the excited luminescent state everywhere outside the local minimum by means of stimulated emission, the luminescence light emitted out of the measurement area afterwards may only stem from the local minimum of the intensity distribution of the luminescence de-excitation light and may thus be assigned to the position of the local minimum within the sample.
In the method known as STED, a very high spatial resolution at a high contrast in imaging a structure of a sample, the structure being marked with a luminophore, is also achieved in practice. Here, however, the luminophore is seriously stressed photochemically and thus tends to bleaching. The reason is that the luminescence de-excitation light, which has to be applied at a high absolute intensity to narrow down the local minimum in the form of a zero point of its intensity distribution, is applied to the luminophore already being in its excited luminescent state. Thus, besides the desired stimulated emission which returns the luminophore into its ground state, other processes, particularly further electronic excitations of the luminophore resulting into bleaching, are also not unlikely. New excitations of the luminophore at first de-excited by stimulated emission may also occur due to the light originally provided for luminescence de-excitation.
A further method of spatial high resolution imaging of a structure of a sample, the structure comprising a luminophore, is known as GSD (Ground State Depletion) scanning fluorescence light microscopy. In this known method, the luminophore, prior to being subjected to the luminescence excitation light, is subjected to luminescence inhibiting light which has an intensity distribution comprising a local minimum. The luminescence inhibiting light transfers the luminophore into a dark state, like for example a long living triplet state, out of which it is not excited into a luminescent state by means of the luminescence excitation light. Everywhere outside the local minimum of the intensity distribution of the luminescence inhibiting light, this transfer into the dark state is saturated. I.e. only in the local minimum of the intensity distribution of the luminescence inhibiting light, the luminophore, after being subjected to the luminescence inhibiting light, is still in its electronic ground state out of which it is excited into the luminescent state by the luminescence excitation light. Luminescence light emitted by the luminophore after excitation by the luminescence excitation light thus stems from the local minimum of the intensity distribution of the luminescence inhibiting light and may thus be assigned to the position of the local minimum within the sample.
In the method known as GSD, there is a considerable danger of bleaching the luminophore as well, because the luminophore, in its long-living dark state, into which it is transferred by the luminescence inhibiting light, has an increased tendency to chemical reactions like, for example, with oxygen, and/or it is exposed to the danger that it is further excited by the luminescence inhibiting light or the luminescence excitation light so that a photochemical bleaching of the luminophore occurs.
A further method of spatial high resolution imaging of a structure of a sample, the structure comprising a luminophore, is known as a variant of RESOLFT (Reversible Saturable Optical Fluorescence Transitions) scanning fluorescence light microscopy which makes use of so-called switchable luminophores. By means of luminescence inhibiting light, these luminophores are switchable out of a first conformation state in which they are acting as luminophores into a second conformation state in which they are not acting as luminophores, i.e. in which they are, at least by means of the luminescence excitation light which is usable for exciting the luminescent state in the first conformation state, not excitable into the luminescent state in which they emit the luminescence light registered as the measurement signal. With a sufficient long lifetime of the second conformation state, only comparatively low light intensities are necessary to saturate this switching everywhere outside a local minimum of the intensity distribution of the luminescence inhibiting light. Further, there is no significant danger that the luminophore transferred into its other conformation state bleaches out of this other conformation state as it does not respond to the luminescence inhibiting light or the luminescence excitation light within this conformation state.
In the practical implementation of RESOLFT scanning fluorescence light microscopy with switchable fluorophores, a spatial resolution and a contrast are observed which lag behind those of STED scanning fluorescence light microscopy. This may be due to the fact that even then when the switching of the switchable luminophore into a conformation state not capable of luminescence is saturated, there is still a noticeable percentage of the luminophore in its conformation state capable of luminescence.
There still is a need of a method of spatial high resolution imaging of a structure of a sample, the structure comprising a luminophore, in which the high spatial resolution and the high contrast of STED scanning fluorescence light microscopy are achieved and in which the luminophore is nevertheless subjected to a lower danger of photochemical bleaching than in all previously known methods of STED scanning fluorescence light microscopy.